Statins, inhibitors of HMG-CoA reductase, exert immunomodulatory effects beyond lipid lowering, yet how they regulate T helper (Th) cell differentiation remains poorly understood. We show that fluvastatin administration to C57BL/6 mice for 7 days suppressed interferon-γ and elevated interleukin-4 production by splenic CD4+ T cells, shifting the immune balance toward Th2 without altering splenocyte numbers or major immune cell subset proportions. This effect was not shared by rosuvastatin, suggesting that lipophilicity-dependent cellular uptake influences immunomodulatory potency. In vitro experiments confirmed a direct Th2-promoting action of fluvastatin on CD4+ T cells. Basophils were excluded as mediating cells, as fluvastatin suppressed rather than enhanced basophil interleukin-4 production. Continuous fluvastatin exposure during granulocyte-macrophage colony-stimulating factor (GM-CSF)-driven dendritic cell (DC) differentiation altered the phenotype of the resulting DCs and markedly enhanced their Th2-polarizing capacity in co-cultures with antigen-specific CD4+ T cells. These functional changes were accompanied by alterations in DC surface molecule expression, although their causal contribution remains to be established. These findings indicate that continuous fluvastatin exposure during DC differentiation is associated with an altered DC phenotype and enhanced Th2-polarizing capacity.
Epithelial-mesenchymal transition (EMT) is crucial for initiation of cancer metastasis. EMT-induced epithelial cancer cells gain stemness characteristics, which enhance cancer malignancy and chemoresistance. Therefore, it is important to clarify the function of EMT-related genes for development of therapeutics for cancer metastasis. Here, we showed that Signal-transducing adaptor protein-2 (STAP-2) inhibits EMT in cancer cells through repressing SMAD-mediated ZEB1 expression, which is a master transcription factor of EMT. STAP-2 inhibits the interaction between SMAD3 and SMAD4, resulting in the downregulation of SMAD2/3/4 complex formation and TGF-β signal-induced gene expression. STAP-2 expression was repressed by SMAD2/3-mediated TGF-β signals and activated by OVOL1, which is one of the EMT repressor transcription factors. Our study showed that STAP-2 is a novel EMT repressor gene which represses ZEB1 expression and motility of cancer cells.
Jasmonate-mediated defense responses redirect plant resources from growth to defense, imposing significant demands on the nitrogen economy to produce nitrogenous compounds, such as alkaloids. However, the mechanisms by which plants maintain essential central metabolic processes, such as nucleotide synthesis, under nitrogen resource competition remain unclear. Therefore, in this study, we characterized the nucleobase cation symporter NtNCS1 in Nicotiana tabacum. Notably, NtNCS1 mRNA exhibited opposing organ-specific regulation by jasmonic acid, with induction in roots (the site of nicotine synthesis) but repression in leaves. NtNCS1 was localized to the plastid envelope in metabolically active cells, including mesophyll, phloem-associated, and, notably, guard cells. This dual regulation suggests that NtNCS1 plays a key role in reallocating nitrogen resources. In roots, its expression possibly contributes to maintaining the essential nucleotide pools for basal metabolism when nitrogen is diverted to alkaloid production. Conversely, its repression in leaves possibly conserves nitrogen by downregulating growth, thereby reallocating resources to defense. Overall, this study highlights the potential roles of NCS1 transporters in safeguarding central metabolism and mediating the plant growth-defense trade-off.
Human norovirus (HuNoV) is a major cause of acute viral gastroenteritis. Although it can be replicated in vitro using human intestinal enteroids (HIEs), generating high-titer viral laboratory stocks remains challenging due to low infection and passage efficiencies. Interferon response and chemokine signaling may be key host pathways that restrict efficient HuNoV replication. Here, we evaluated the effect of their inhibitors, ruxolitinib-a Janus kinase (JAK) 1/JAK2 inhibitor-and TAK-779-a C-X-C motif chemokine receptor 3 (CXCR3)/C-C motif chemokine receptor (CCR) 5/CCR2 antagonist-on HuNoV replication in wild-type jejunal HIEs. TAK-779 increased GII.17 HuNoV replication by 2.3- and 6.0-fold at 48 and 96 h postinfection (hpi), respectively, compared with the DMSO-treated control. In contrast, ruxolitinib alone did not affect viral replication. Notably, simultaneous treatment with these compounds further enhanced GII.17 replication by 6.0- and 10.7-fold at 48 and 96 hpi, respectively, which enabled serial passaging. These findings imply that the simultaneous suppression of these pathways potentially benefits GII.17 HuNoV replication.
Using a genetic screening approach based on an inducible gene-activating system and cell sorting, Down syndrome critical region 3 (DSCR3) was isolated as a gene whose overexpression increased cell size. Fibroblasts derived from individuals with Down syndrome (DS) exhibit elevated DSCR3 expression at both the mRNA and protein levels, correlating with increased cell volume compared to fibroblasts from healthy donors. Despite a slower proliferation rate, DS fibroblasts demonstrate higher basal and maximal mitochondrial respiration, suggesting enhanced metabolic activity associated with increased cell size. siRNA-mediated knockdown of DSCR3 reduces cell size in both DS and normal fibroblasts, indicating its general role in cell size regulation. As DSCR3 is a component of the retriever complex involved in endosomal cargo recycling, these findings position membrane protein trafficking as a novel module for cell size control.
Brg1 is a core ATPase subunit of the SWItch/Sucrose Non-Fermentable (SWI/SNF) chromatin-remodeling complex that regulates DNA accessibility for RNA polymerase II (Pol II), transcription factors, and DNA repair enzymes. Phosphoproteomic profiling of highly malignant ovarian clear cell carcinoma (OCCC) cell lines revealed reduced levels of several SWI/SNF components and decreased Brg1 phosphorylation within its histone-binding region. Prior work showed that phosphorylation-mimic and phosphorylation-deficient mutants alter chromatin-silencing factors, producing chromatin condensation and decondensation, respectively. Here, we performed RNA-seq in Brg1-deficient JHOC-5 cells and in isogenic cells expressing Brg1-WT, phosphorylation-mimic brg1-S1452D, or phosphorylation-deficient brg1-S1452A. PCA and hierarchical clustering separated brg1-S1452A from the other lines, indicating a strong transcriptional impact of Ser1452 dephosphorylation. JHOC-5 and brg1-S1452A shared altered expression of Pol II-promoter-regulated genes. Apoptosis genes (BCL2, FGFR2, ZC3H12A, and NFKBIA) showed reciprocal expression between brg1-S1452D and brg1-S1452A, consistent with increased and decreased apoptosis, respectively. Genes linked to cell adhesion/migration and neuronal development also differed, accompanied by reduced cell circularity in brg1-S1452A. Overall, Brg1 phosphorylation at Ser1452 shapes SWI/SNF-mediated chromatin remodeling to regulate transcriptional programs controlling apoptosis and cell morphology.
Signal transducer and activator of transcription 3 (STAT3) is essential for embryonic stem (ES) cell self-renewal, and its mutations are found in various human diseases. In this study, we investigated the effects of disease-associated STAT3 mutations (Y640F and G656insF) on ES cell properties. These mutations maintained ES cell self-renewal in the absence of LIF and MEK/GSK3 inhibitors. Although these mutants exhibited enhanced transcriptional activity, their phosphorylation status remained unchanged after LIF stimulation. Importantly, these mutations functioned independently of endogenous STAT3, as demonstrated in STAT3 knockout ES cells. Our findings reveal that disease-associated STAT3 mutations can maintain stem cell properties through phosphorylation-independent mechanisms, providing insights into how these mutations might regulate stem cell functions in both developmental and pathological contexts.
Cancer cells proliferate uncontrollably, and a major challenge in cancer research is to identify strategies that selectively eliminate cancer cells while sparing normal cells. Squamous cell carcinoma antigen recognized by T cells 1 (SART1) was originally identified as a carcinoma-associated antigen and is frequently overexpressed in cancer cells. Recently, SART1 has been identified as a microtubule-associated protein required for spindle pole formation and cell division. Here, we show that partial depletion of SART1 by RNA interference selectively disrupts mitotic spindle assembly and induces cell death in cancer cells. siRNA-mediated knockdown reduced SART1 protein levels comparably in multiple human cell types, yet spindle defects and apoptotic cell death were observed in HeLa and U2OS cancer cells but not in non-transformed BJ fibroblasts or RPE1 epithelial cells. Consistent with this cancer-selective sensitivity, SART1 depletion markedly suppressed oncogene c-Myc-induced transformation of RPE1 cells without impairing the growth of untransformed cells. These results demonstrate that partial depletion of SART1 preferentially induces spindle defects and cell death in transformed cells. Given that complete loss of SART1 is incompatible with normal development, our findings suggest that transient or partial inhibition of SART1 may provide a basis for selectively eliminating transformed cells while sparing normal cells.
SQSTM1 is one of the causative genes of neurodegenerative disorders, amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The SQSTM1 protein regulates the degradation of polyubiquitinated proteins and autophagosome formation through its interaction with microtubule-associated protein light chain 3 (MAP1LC3/LC3). However, the molecular mechanisms by which SQSTM1-LC3 binding regulates the autophagy-endolysosomal system (APELS) remain unclear. To elucidate the spatiotemporal role of SQSTM1, we transiently expressed wild-type SQSTM1 or missense mutants carrying mutations in the LC3-interacting region (LIR), fused with the photoconvertible fluorescent protein Dendra2. Live-cell fluorescence imaging and co-localization analyses with markers of the APELS were then performed. Particle analysis of photoconverted or non-photoconverted SQSTM1-positive structures in live cells revealed that the pathogenic L341V variant formed larger structures than the wild-type. Co-localization analyses further showed that both the L341V and artificial LIR3A mutants accumulated in large ubiquitin-positive structures, likely due to impaired localization to autophagosomes. These results suggest that mutations within the LIR differentially affect autophagosome formation and cargo degradation within APELS-related compartments, highlighting the importance of SQSTM1 structural integrity in ALS/FTD pathogenesis.
The noncanonical NF-κB pathway regulates immune development and inflammation through RelB nuclear translocation, yet the dynamics of this process at the single-cell level remain poorly understood. Using live-cell imaging of RelB-Venus knock-in mouse embryonic fibroblasts, we show that LTβR or TWEAK stimulation induces four distinct RelB nuclear translocation patterns: oscillating, prolonged activation, transient activation, and non-responding. Approximately 40% of cells exhibited oscillatory behavior with a predominant period of 1.5-2.0 h, similar to RelA oscillations in the canonical pathway. Mechanistically, RelB oscillations required CRM1-mediated nuclear export, ongoing protein synthesis, NIK-dependent noncanonical signaling, and the IκBα nuclear export signal. Knockdown or knockout of Nfkb2 (encoding p100) induced spontaneous RelB oscillations without stimulation, identifying p100 as a threshold regulator that controls oscillation probability through cytoplasmic sequestration of RelB. Co-immunoprecipitation analysis revealed dynamic RelB-IκBα complex formation in both cytoplasmic and nuclear fractions following LTβR stimulation. Furthermore, disruption of RelB oscillations in NfkbiaNES/NES cells was associated with impaired induction of NF-κB target genes. These findings provide the first experimental characterization of RelB oscillatory dynamics and reveal both conserved and pathway-specific mechanisms governing noncanonical NF-κB signaling.
Carcinoma-associated fibroblasts (CAFs), frequently present in the stroma of human breast carcinomas, influence tumor characteristics. A forkhead box protein O1 (FOXO1) transcription factor is a critical mediator of the cellular responses to oxidative stress in various cell types. However, the roles of FOXO1 in CAFs have been poorly understood. Here, we show more intense FOXO1 staining in stromal fibroblasts in human breast cancers compared to those in non-cancerous regions. Notably, stronger stromal FOXO1 staining is significantly associated with poorer outcomes in 237 breast cancer patients. FOXO1 expression is also more abundant in cultured human breast CAFs compared to the control counterpart human mammary fibroblasts. FOXO1 expression is upregulated in control fibroblasts by malnutrition and hypoxia, suggesting such starvation to mediate increased FOXO1 expression in CAFs. Of note, treatment with AS1842856, a FOXO1 inhibitor, significantly attenuates growth and viability in CAFs relative to control fibroblasts. Suppression of FOXO1 expression by shRNA also substantially inhibits CAFs' growth in culture. When the FOXO1-knockdowned CAFs were implanted with breast cancer cells into recipient mice, the number of these fibroblasts present in developed tumors tends to decrease. Taken together, these findings indicate that FOXO1 expression in human breast CAFs is required for their growth and viability.
In mammalian sex differentiation, Sox9 expression in undifferentiated gonads determines male development. Sox9 is regulated by multiple enhancers, of which Enh13/mXYSRa is the most critical. Disruption of the GATA4 binding site in Enh13/mXYSRa causes XY sex reversal in the C57BL/6J (B6J) strain, but not in the mixed genetic background of B6J × DBA/2. To clarify the cause of these differences, mice with the same mutation were generated in DBA/2. Homozygous mutants developed as XY males. We hypothesized that the phenotypic differences between strains were due to functional differences of another enhancer. Mice lacking TESCO and carrying a mutation in the GATA4 binding site were generated and analyzed. The mutants developed according to their chromosomal sex despite reduced Sox9 expression. These findings imply that the DBA/2 genetic background contains factors preventing Sox9 expression from falling below the threshold required for male development. Bulk RNA-seq analysis of fetal gonads during sex determination identified strain-dependent differences in intermediate mesoderm development and protease inhibition pathways; the study also revealed that the expression of Sox8 has a redundant role in Sertoli cell differentiation to that of Sox9 in male embryonic gonads. These differences may contribute to the phenotypic variation observed between strains.
The lymphocyte antigen-6 (Ly6)/urokinase-type plasminogen activator receptor (uPAR) superfamily (LU superfamily) of proteins is involved in diverse biological processes. In Drosophila melanogaster, members of the LU superfamily have undergone lineage-specific gene duplication and acquired specialized functions in distinct tissues. A glycosylphosphatidylinositol (GPI)-anchored LU family protein Belly roll (Bero) has recently been shown to regulate larval escape behavior; however, its cellular expression profile and potential roles remain incompletely understood. In this study, we generated a bero-GAL4 T2A transgenic line to delineate endogenous bero expression. This analysis revealed that bero is expressed in the peptidergic neurons in the central nervous system (CNS) that had not been documented in previous studies, as well as in the peripheral nervous system (PNS) and non-neuronal tissues, such as the anal pad and epidermis. Reanalysis of publicly available single-cell RNA sequencing (scRNA-seq) datasets demonstrated that bero is expressed in several peptidergic neurons. These findings suggest that Bero is specifically expressed in diverse peptidergic neurons and may play important roles in coordinating hormonal and neural regulation in D. melanogaster.
Sema3c is specifically expressed in the cardiac outflow tract (OFT) of the developing mouse heart and has been implicated in OFT polarization and great artery formation. However, the regulatory basis underlying its spatially restricted expression remains unclear. To investigate the mechanisms underlying OFT-specific Sema3c expression, we utilized chromatin accessibility data from distinct segments of the developing heart and identified a differentially accessible region as an OFT-specific Sema3c enhancer candidate. Unlike previously characterized Sema3c enhancers, this region is located distal to the transcription start site. Reporter analysis using transgenic mouse embryos demonstrated that this region exhibits transcriptional activity from E8.5 onward and remains specifically active in the OFT myocardium throughout heart development. We further defined a minimal 603 bp enhancer whose activity depends on GATA binding sites. This enhancer provides insight into the mechanisms underlying spatially restricted Sema3c expression involved in OFT development.
Chromatin organization changes during aging, accompanied by alterations in higher-order chromosome architecture, contributing to the acquisition of senescent cell-specific functions. Recent studies have revealed that chromatin states and their regulatory mechanisms vary between individual cells. However, analyses of higher-order chromosome structure have been performed mostly at the population level. Therefore, approaches capable of directly quantifying such alterations at the single-cell level are required. Here, we applied persistent homology (PH), a topological data analysis framework, to quantify spatial features of γH2AX foci and DAPI-stained chromatin in single-cell fluorescence images. Using a model of pathological senescence induced by impaired acetylation-dependent histone H2AX exchange, we extracted topological features followed by machine learning analysis. Unsupervised analysis revealed spatial patterns associated with pathological senescence in both γH2AX and DAPI signals. Notably, classifications derived from γH2AX foci showed correspondence with those from DAPI-based chromatin organization, suggesting that local DNA damage patterns reflect global chromatin structure. Supervised models further distinguished normal and pathological senescent cells, with DAPI-derived features highlighting the contribution of nuclear intensity gradients. These findings demonstrate that PH enables quantitative characterization of nuclear architecture at the single-cell level and provides a framework for dissecting chromatin reorganization during cell state transitions.
Homology-directed repair (HDR)-mediated knock-in efficiency is a composite of double-strand break (DSB) induction and repair pathway selection during targeted insertion. Thus, optimization of HDR-mediated knock-in presents significant challenges. To address this, we employed a mouse embryonic stem cell-based triple-reporter platform to examine how donor design and experimental parameters are associated with DSB induction and the balance between HDR-mediated knock-in and end-joining-mediated targeted insertion (EJ-TI). Our analysis reveals that donor design directly impacts DSB induction: co-electroporation of donor plasmids with Cas9/gRNA reduced cleavage efficiency, and Homology-Independent Targeted Integration (HITI) sequences further reduced this efficiency, thereby biasing overall knock-in efficiency. When normalized to DSB levels, HITI increased total targeted insertion yield (knock-in + EJ-TI) with shorter arms and favored HDR-mediated knock-in over EJ-TI with longer arms. Conversely, under adeno-associated virus (AAV) donor conditions, HITI showed donor-format dependency. It increased knock-in and decreased EJ-TI only with a self-complementary AAV donor, but had little effect with a single-stranded AAV donor. These results highlight the importance of separately evaluating DSB induction, total targeted insertion yield, and the balance between HDR-mediated knock-in and EJ-TI using a triple-reporter platform when optimizing knock-in strategies.
The Rec8 cohesin complex is required for the pairing and recombination of homologous chromosomes during meiosis, as well as for the cohesion of sister chromatids. In the fission yeast Schizosaccharomyces pombe, we previously identified a rec8-F204S mutant that lost the ability to assemble the axis-loop chromatin structure without losing sister chromatid cohesion. This mutant showed reduced meiotic recombination, indicating that pairing and recombination of homologous chromosomes require the formation of the axis-loop chromatin structure mediated by the Rec8 cohesin complex. Loading of the Rec8 cohesin complex onto chromatin is mediated by Mis4 (NIPBL in humans; Scc2 in yeast). In this study, to elucidate the functions of Mis4, we identified a mis4-LR mutant (L1150S and R1159G) that reproduced the phenotypes of the rec8-F204S mutant, which is defective in chromatin axis formation and homologous recombination while retaining sister chromatid cohesion. These mutation sites (Mis4-L1150, Mis4-R1159, and Rec8-F204) are all localized at the interaction surface between Mis4 and Rec8. Biochemical analysis revealed that the Mis4-LR mutant protein exhibited reduced Rec8-binding activity. Considering that the mis4-LR mutant phenocopied the rec8-F204S mutant, our results demonstrate that the Mis4-Rec8 interaction is required for proper formation of Rec8-dependent meiotic chromosome axis.
Circulating microRNAs (miRNAs) are promising biomarkers and many studies have been reported. However, there are discrepancies between studies due to differences in the inclusion criteria, experimental procedures, and serum/plasma processing conditions. In this study, we aimed to develop a comprehensive set of miRNA markers to assess the serum quality, which fluctuates depending on the storage duration after blood collection. This study was designed to identify circulating miRNAs that were unstable when stored at room temperature (23°C-27°C) for more than 0.5-1 h after centrifugation to separate the serum. The results showed that 17 miRNAs were more than 5% variable in their signals after storage at room temperature for more than 0.5 h. Of these, seven miRNAs were identified whose signals changed significantly by more than 1 in signal level after 1 h of storage at room temperature, showing time-dependent fluctuations in detection levels. These miRNAs were further evaluated between the two clinical sample groups with different preprocessing times. miR-4429 and miR-6511b-5p showed a significant decrease in signals in the sample group with longer processing times. These miRNAs can be used as markers to assess sample quality and reflect the time-dependent storage conditions of clinical samples.
Mitotic chromosomes, consisting of a pair of rod-shaped chromatids, emerge from a dramatic reorganization of nucleosome fibers. However, the mechanisms by which local chromatin organization influences large-scale mitotic chromatid architecture remain poorly understood. Here, we report a modified cell-free assay in which erythrocyte nuclei, instead of conventionally used sperm nuclei, are incubated in Xenopus mitotic egg extracts. This modification enables mitotic chromatids to assemble from substrates containing regularly spaced, dense nucleosome arrays, allowing chromatid formation to be experimentally separated from nucleosome assembly. In this system, depletion of the linker histone H1.8 results in thinner chromatids with enhanced individualization, whereas excess H1.8 loading promotes chromatid clustering through its C-terminal tail. Chromatid clustering is also observed upon depletion of the histone chaperone Nap1 or the chromatin remodeler ISWI, whose roles have been underappreciated in conventional assays. Together, our findings demonstrate that histone density and dynamics cooperate with condensins and topoisomerase IIα to shape mitotic chromatid architecture.
The transcription factor E2F plays crucial roles in cell proliferation and tumor suppression. In the resting state of normal cells, E2F activity is suppressed by binding of the tumor suppressor pRB and its family members. E2F activated by growth stimulation under the control of pRB (physiological E2F) activates growth-related genes and facilitates cell proliferation. In contrast, E2F activated by loss of pRB control, such as forced inactivation of pRB (distinct E2F), activates tumor suppressor genes such as ARF and TAp73 to suppress tumorigenesis. We previously reported that these genes are specifically activated by distinct E2F but not by physiological E2F. In almost all cancers, pRB function is disabled, generating distinct E2F activity, which is tolerated due to concomitant dysfunction of the tumor suppressor p53. Hence, distinct E2F activity is a characteristic feature of cancer cells. Mouse embryonic stem cells (mESCs) proliferate rapidly with shortened gap phases, enhanced cyclin dependent kinase (CDK) activity and hyper-phosphorylated (inactive) pRB. This observation prompted us to examine whether mESCs possess distinct E2F activity. We show here that, like cancer cell lines, mESCs exhibit distinct E2F activity. Moreover, introduction of a small amount of CDK inhibitors enhanced deregulated E2F activity, suggesting that it is suppressed by CDK activity in mESCs.
